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United States Patent | 5,081,033 |
Dorn ,   et al. | January 14, 1992 |
Methods and compositions are disclosed for the rapid identification of fungal pathogens. In a preferred method, a sample of human body fluid is obtained and subjected to analysis comprising inoculating a nutritive medium, incubating said inoculum for such time and temperature as is necessary for visible fungal colony formation, thereafter plating a sample of said visible fungi on miniature culture plates for example having the dimensions of about 33.times.75.times.5 millimeters, said plates containing differential fungal media, and thereafter identifying funal pathogens in a manner consistent with said medium selected. In a related method utilizing the differential carbohydrate assimilation properties of fungal pathogens, a sample of human body fluid is obtained and subjected to analysis comprising inoculating a nutritive media, incubating said inoculum for such time and temperature as is necessary for visible fungal colony formation, thereafter inoculating a carbohydrate depleted medium with a sample of said visible fungi, incubating said inoculum for a period of time, thereafter plating a sample of said inoculated carbohydrate depleted medium on miniature culture plates, said plates containing differential carbohydrate assimilation media, and thereafter identifying said fungal pathogens in a manner consistent with said medium selected. Novel compositions useful in practicing the disclosed methods are provided. These include an improved fungal growth medium, the improvement comprising the novel addition of ammonium ions to a medium comprising purified saponin, oxgall, a substrate for phenol oxidase and a supporting agent.
Inventors: | Dorn; Gordon L. (Dallas, TX); Fleming; William H. (Richardson, TX); Knezek; Karen L. (Garland, TX) |
Assignee: | Wadley Technologies, Inc. (Dallas, TX) |
Appl. No.: | 885201 |
Filed: | July 14, 1986 |
Current U.S. Class: | 435/244; 435/34; 435/255.3; 435/255.4; 435/255.7; 435/922; 435/924 |
Intern'l Class: | C12N 001/38; C12N 001/14; C12Q 001/04 |
Field of Search: | 435/244,34,254,922,924 |
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3728228 | Apr., 1973 | Duranty | 195/127. |
3870600 | Mar., 1975 | Youssef | 195/81. |
3875015 | Apr., 1975 | Wadley et al. | 195/127. |
3883398 | May., 1975 | Ono | 195/127. |
3883425 | May., 1975 | Dorn | 210/23. |
3928136 | Dec., 1975 | Launey | 195/54. |
4030980 | Jun., 1977 | Beckford et al. | 195/139. |
4144133 | Mar., 1979 | Dorn et al. | 195/100. |
4179266 | Dec., 1979 | Lukacsek | 435/254. |
Foreign Patent Documents | |||
515954 | May., 1981 | AU. | |
2401222 | Mar., 1979 | FR. | |
1132455 | Feb., 1966 | GB. | |
1026253 | Apr., 1966 | GB. | |
1535643 | Dec., 1978 | GB. |
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TABLE 1 ______________________________________ COMPARISON OF THE MINIATURIZED SOC PLATE WITH OTHER AVAILABLE SYSTEMS FOR GERM TUBE FORMATION BY Candida albicans Germ Tube Formation No. of + .+-. Clumping System Isolates No. % No. % No. % ______________________________________ 1. SOC 20 20 100 0 0 0 0 2. Egg white 20 20 100 0 0 0 0 3. Fetal bovine 20 20 100 0 0 2 10 serum 4. Flow GBE 20 18 90 2 10 4 20 tube 5. API GT 20 17 85 3 15 16 80 Microtest ______________________________________
TABLE 2 __________________________________________________________________________ MORPHOLOGY AND PIGMENT PRODUCTION ON MINIATURIZED SOC MEDIUM AT VARIOUS CONCENTRATIONS OF CAFFEIC ACID Weight Percent Caffeic Acid Organism Incubation Time (hrs.) 0.006 0.06 0.12 0.6 __________________________________________________________________________ Candida albicans 3 gt+ gt+ gt-+ gt+- 24 fil +/cm+ fil +/cm+ fil +/cm+ fil -/cm- Candida stellatoidea 3 gt- gt- gt- gt- 24 fil +/cm- fil +/cm- fil +/cm- fil -/cm- Candida tropicalis 3 ps+ ps+ ps- ps- 24 fil+ fil+ fil+ fil- Candida krusei 3 b+ b+ b+ b+ 24 fil+ fil+ fil+ fil- Candida guilliermondii 3 b+ b+ b+ b+ 24 fil+ fil+ fil+ fil- Candida parapsilosis 3 b+ b+ b+ b+ 24 fil+ fil+ fil+ fil- Cryptococcus neoformans* 18 brn brn brn brn Cryptococcus laurentii 18 crm crm crm crm Cryptococcus albidus 18 crm crm crm crm Cryptococcus diffluens 18 crm crm crm crm __________________________________________________________________________ *Twenty (20) strains were tested gt = germ tube; fil = filament; cm = chlamydospore, ps = pseudohyphae b = blastospore, brn = brown, crm = cream
TABLE 3 ______________________________________ EFFECTS ON NH.sub.4 Cl ADDITION TO SOC ON CHLAMYDOSPORE PRODUCTION IN TWENTY STRAINS OF Candida albicans Chlamydospore Production NH.sub.4 Cl, Molarity 0 0.001 0.01 0.1 Incubation No. of % % % % Time (hrs.) Strains Positive Positive Positive Positive ______________________________________ 24 20 45.0 80.0 100.0 50.0 48 20 60.0 100.0 100.0 80.0 72 20 100.0 100.0 100.0 100.0 ______________________________________
TABLE 4A __________________________________________________________________________ EXAMINATION OF THE MINIATURE SUCROSE ASSIMILATION PLATE WITH VARIOUS YEASTS Sucrose Assimilation 5 hr. 24 hr. No. of Correct + - + - Organism Isolates Response No. % No. % No. % No. % __________________________________________________________________________ C. albicans 120 + 120 100.0 0 0.0 120 100.0 0 0.0 C. stellatoidea 21 - 0 0.0 21 100.0 0 0.0 21 100.0 C. tropicalis 29 + 29 100.0 0 0.0 29 100.0 0 0.0 C. parapsilosis 31 + 0 0.0 31 100.0 31 100.0 0 0.0 C. krusei 18 - 0 0.0 18 100.0 0 0.0 18 100.0 C. glabrata 34 - 0 0.0 34 100.0 0 0.0 34 100.0 __________________________________________________________________________
TABLE 4B __________________________________________________________________________ EXAMINATION OF MINIATURE CARBOHYDRATE ASSIMILATION PLATES WITH VARIOUS YEASTS Assimilation 5 hr. 24 hr. No. of Correct + - + - Organism Isolates Carbohydrate* Response No. % No. % No. % No. % __________________________________________________________________________ C. albicans 3 suc + 3 100.0 0 0.0 3 100.0 0 0.0 malt + 3 100.0 0 0.0 3 100.0 0 0.0 galac + 3 100.0 0 0.0 3 100.0 0 0.0 treh + 2 66.7 1 33.3 3 100.0 0 0.0 lac - 0 0.0 3 100.0 0 0.0 3 100.0 C. stellatoidea 3 suc - 0 0.0 3 100.0 0 0.0 3 100.0 malt + 3 100.0 0 0.0 3 100.0 0 0.0 galac + 3 100.0 0 0.0 3 100.0 0 0.0 treh + 0 0.0 3 100.0 3 100.0 0 0.0 lac - 0 0.0 3 100.0 0 0.0 3 100.0 C. tropicalis 3 suc + 3 100.0 0 0.0 3 100.0 0 0.0 malt + 3 100.0 0 0.0 3 100.0 0 0.0 galac + 1 33.3 2 66.7 3 100.0 0 0.0 treh + 1 33.3 2 66.7 3 100.0 0 0.0 lac - 0 0.0 3 100.0 0 0.0 3 100.0 C. parapsilosis 3 suc + 3 100.0 0 0.0 3 100.0 0 0.0 malt + 1 33.3 2 66.7 3 100.0 0 0.0 galac + 2 66.7 1 33.3 3 100.0 0 0.0 treh + 0 0.0 3 100.0 3 100.0 0 0.0 lac - 0 0.0 3 100.0 0 0.0 3 100.0 __________________________________________________________________________ *suc = sucrose; malt = maltose; galac = galactose; treh = trehalose; lac = lactose
TABLE 5 ______________________________________ EXAMINATION OF THE MINIATURE UREASE PLATE WITH VARIOUS YEASTS Urease Hydrolysis* No. of Correct + - Organism Isolates Response No. % No. % ______________________________________ Cr. neoformans 81 + 81 100.00 0 0.0 Cr. albidus 20 + 20 100.0 0 0.0 Cr. laurentii 10 + 10 100.00 0 0.0 Cr. diffluens 24 + 24 100.00 0 0.0 C. albicans 122 - 0 0.0 122 100.0 C. stellatoidea 21 - 0 0.0 21 100.0 C. tropicalis 29 - 0 0.0 29 100.0 C. parapsilosis 32 - 0 0.0 32 100.0 C. krusei 18 -(+)** 0 0.0 18 100.0 C. glabrata 34 - 0 0.0 34 100.0 ______________________________________ *The reactions were read at one hour and negative reactions were reconfirmed at 24 hours. **C. krusei has been reported to rarely show a positive urease reaction, but the strains used in this experiment were negative.
TABLE 6A __________________________________________________________________________ COMPARISON OF MICROTITER .TM. CARBOHYDRATE AGAR ASSIMILATION WITH REPORTS FROM CONVENTIONAL ASSIMILATION METHODS* No. of Assimilation at 24 hrs.** Organism Isolates System dextrose galactose sucrose maltose cellibiose trehalose lactose raffinose __________________________________________________________________________ C. albicans -- Conventional + + + + - + - - 45 Microtiter + + + + - + - - C. stellatoidea -- Conventional + + - + - + - - 12 Microtiter + + - + - + - - C. tropicalis -- Conventional + + + + v + - - 32 Microtiter + + + + .-+. + - - C. parapsilosis -- Conventional + + + + - + - - 12 Microtiter + + + + - + - - C. krusei -- Conventional + - - - - - - - 8 Microtiter + - - - - - - - C. glabrata -- Conventional + - - - - + - - 16 Microtiter + - - - - + - - Cr. neoformans -- Conventional + + + + w + - w 8 Microtiter + + + + .-+. .+-. - - Cr. laurentii -- Conventional + + + + + + + + 2 Microtiter + + + + + + + + Cr. albidus -- Conventional + w + + + + w w var. diffluens 7 Microtiter + .-+. + + .+-. + - - __________________________________________________________________________ *Conventional Methods: Wickerham liquid method, Saito agar method, auxanographic method. **v = variable; w = weak; .+-. = .gtoreq.50%; .-+. = .ltoreq.50%
TABLE 6B __________________________________________________________________________ COMPARISON OF MICROTITER .TM. CARBOHYDRATE AGAR ASSIMILATION WITH A.P.I. 20C STRIP REPORTS Total No. of Assimilation at 24 hrs. Iso- dextrose galactose sucrose maltose cellibiose trehalose lactose raffinose Organism lates System No. % No. % No. % No. % No. % No. % No. % No. % __________________________________________________________________________ C. albicans --* API -- 100 -- 100 -- 96 -- 100 -- 0 -- 99 -- 0 -- 0 45 Micro- 45 100 45 100 45 100 45 100 0 0 45 100 0 0 0 0 titer C. stellatoidea -- API -- 100 -- 100 -- 0 -- 100 -- 0 -- 1 -- 0 -- 0 12 Micro- 12 100 12 100 0 0 12 100 0 0 12 100 0 0 0 0 titer C. tropicalis -- API -- 100 -- 99 -- 100 -- 100 -- 12 -- 100 -- 0 -- 0 32 Micro- 32 100 32 100 32 100 31 97 15 47 32 100 0 0 0 0 titer C. parapsilosis -- API -- 100 -- 100 -- 100 -- 100 -- 0 -- 97 -- 0 -- 0 12 Micro- 12 100 12 100 12 100 12 100 0 0 12 100 0 0 0 0 titer C. krusei -- API -- 100 -- 0 -- 0 -- 0 -- 0 -- 0 -- 0 -- 0 8 Micro- 8 100 0 0 0 0 0 0 0 0 0 0 0 0 0 0 titer C. glabrata -- API -- 100 -- 0 -- 0 -- 0 -- 0 -- 99 -- 0 -- 0 16 Micro- 16 100 0 0 0 0 0 0 0 0 16 100 0 0 0 0 titer Cr. neo- -- API -- 100 -- 99 -- 100 -- 100 -- 37 -- 73 -- 0 -- 86 formans 8 Micro- 8 100 8 100 8 100 8 100 1 13 4 50 0 0 0 0 titer Cr. laurentii -- API -- 100 -- 100 -- 100 -- 92 -- 92 -- 92 -- 99 -- 99 2 Micro- 2 100 2 100 2 100 2 100 2 100 2 100 2 100 2 100 titer Cr. albidus -- API -- 100 -- 0 -- 100 -- 99 -- 99 -- 93 -- 0 -- 68 var. diffluens 7 Micro- 7 100 2 29 7 100 7 100 6 86 7 100 0 0 0 0 titer __________________________________________________________________________ *Total number of isolates given only in A.P.I. databank.